The protozoa of the class Sporozoea include sporocyst-forming protozoa, many of which are etiologic in a myriad of diseases of animals and people around the world. Of particular commercial concern are the diseases caused by Sporozoea of the subclass Coccidia, order Eucoccidiida and suborder Eimeriina, as well as the sporocyst-forming protozoa of the genera Cystoisospora, Eimeria, Isospora, Neospora, Sarcocystis, Toxoplasma, Tyzzeria and Wenyonella.
In particular, coccidiosis is an enteric disease of animals, including domestic livestock e.g. poultry, caused by protozoan parasites of the subclass Coccidia, genus Eimeria. These are obligate intracellular protozoan parasites that replicate primarily in the intestinal epithelium. These parasites have a monoxenous life cycle and exhibit a high degree of host-species and tissue specificity. Overall, the combination of losses due to coccidiosis and the costs of prophylactic medication results in significant annual economic losses to the affected industries.
The economic effects of coccidiosis infections in domestic animals is especially severe in the poultry industry, where intensive housing of birds favors the spread of the disease. For fowl, e.g., poultry, and other domestic birds, Coccidia infection results in economic loss from stunting and skin discoloration. In addition, numerous Eimeria species can infect a single host via the oral route and/or by inhalation of the infectious particles from the environment. Once ingested, the parasite penetrates and damages the mucosal layer of the intestinal wall, causing acute morbidity, e.g, resulting in decreased growth and feed utilization in the infected avians.
Protozoa of the genera Eimeria, Isospora, Cystoisospora, or Cryptosporidium typically only require a single host to complete their life cycle. Under natural conditions, the life cycle begins with the ingestion of sporulated oocysts from the environment. The cell wall of the ingested sporulated oocyst is broken by mechanical disruption in the gizzard and/or digested by intestinal enzymes. Within the sporocysts are the sporozoites, which are the infective stage of the organism. The sporozoites invade the intestinal mucosa, or epithelial cells in other locations. The site of infection is characteristic of the species involved.
Within a host animal's cells, sporozoites develop into multinucleate meronts, also called schizonts. Each meront nucleus develops into an infective body called a merozoite, that enters new cells and repeats the process. After a variable number of asexual generations, merozoites develop into either microgametocytes or macrogametes. Microgametocytes develop into many microgametes which, in turn, fertilize the macrogametes. The resulting zygotes encyste by forming a tough outer coat, and are then called oocysts, which are shed unsporulated in the feces. Under proper environmental conditions, the oocysts sporulate and become infective. Ingestion by susceptible animals then repeats the cycle.
Infection with Coccidia elicits a species-specific immunity. For example, there are seven known species of Coccidia which infect chickens, six of which are considered to be moderately to severely pathogenic.
Thus, in domestic avians, the introduction of another species of Coccidia or the introduction of new, previously unexposed birds may result in an outbreak of the disease. Oocysts are resistant to extremes in pH, detergents, proteolytic, glycolytic, and lipolytic enzymes, mechanical disruption (although they can be broken by laboratory methods) and chemicals such as sodium hypochlorite and potassium dichromate, and will survive for many weeks outside the host. For example, there are seven known species of Coccidia which infect chickens, six of which are considered to be moderately to severely pathogenic.
Numerous methods have been developed to immunize poultry against Coccidia. All commercially successful methods are based on the administration of live encysted protozoa, often of an attenuated strain. The most common route of inoculation is oral, although other routes have been used. Edgar, U.S. Pat. No. 3,147,186, teaches vaccination of chickens by oral administration of viable E. tenella sporulated oocysts. Davis et al., U.S. Pat. No. 4,544,548, teaches a method of vaccination by continuous administration of low numbers of sporulated oocysts, with or without simultaneous administration of anti-Coccidial drugs.
Attenuation can be achieved by serial passage in chickens. Selection of strains of Coccidia showing early formation of oocysts, and attenuation, has been described by 5,055,292, incorporated by reference herein its entirety.
Orally-administered attenuated strains of sporocysts have also been utilized to confer immunity against coccidiosis. [Shirley, U.S. Pat. No. 4,438,097; McDonald, U.S. Pat. No. 5,055,292; and Schmatz et al., PCT publication No. WO 94/16725]. An alternative to attenuation is disclosed in Jenkins et al., Avian Dis., 37(1):74-82 (1993), which teaches the oral administration of sporozoites that have been treated with gamma radiation to prevent merogony.
Parenteral routes of vaccination have included subcutaneous or intraperitoneal injection of excysted sporozoites, Bhogal, U.S. Pat. No. 4,808,404; Bhogal et al., U.S. Pat. No. 5,068,104, and intra-ovo injection of either oocysts or sporocysts, Evans et al., PCT publication No. WO 96/40233; Watkins et al., Poul. Sci., 4(10):1597-602 (1995). Thaxton, U.S. Pat. No. 5,311,841, teaches a method of vaccination against Coccidia by administration of oocysts or sporozoites to newly hatched chicks by yolk sac injection.
The need for viable protozoa for vaccination is a common factor in all of the current vaccination methods. Non-viable protozoa, or antigens from protozoa, have been unsuccessful in conferring the high level of immunity needed to protect against clinical infection.
Heretofore, viable, living protozoa, e.g. oocysts, have previously been harvested from living, intact infected avians. However, the use of live animals or, in some Coccidia species, chick eggs, to produce viable Coccidia cells for vaccine production is both costly and inefficient.
An economical and efficient alternative method for producing the required quantities of Coccidia for use in vaccine production, and other purposes, has long been sought. Propogation of Coccidia in vitro has been attempted, but with only limited success. Madin Darby Bovine Kidney (MDBK) cells have been reported to support Eimeria growth in vitro but the development is limited to one replication cycle of asexual development [D. M. Schmatz, Adv, Cell Culture, 5:241 (1987)]. Eimeria oocysts from E. acervulina [M. Nacri-Bontemps, Ann. rech. Vet., 7:223 (1976)] and E meleagrimitis [Augustin et al, J. Protozool., 25:82, (1978)] as well as E bovis [Speer et al, Z. Parasitenkd, (1973)], were obtained when initial host-derived merozoites have been used as the inoculum. However, as noted above, the requirement to use merizoites derived from infected animal tissues limits the usefulness of these approaches.
U.S. Pat. No. 5,846,527 describes an avian cell line derived from abnormal embryonic tissue that supports the growth of E. tenella and E. necatrix, but replication was sustained for only 72 hours. Another drawback is that an immortalized cell line derived from chickens provides a risk for transmitting tumorigenicity to chickens if used to make live vaccines.
Limited passage of merozoites has been known. For example, in U.S. Pat. No. 6,500,438, sporozoites were infected into primary chick kidney cells (PCK) that are grown in culture as cell aggregates, using a modification of the method described in D. J. Doran, J. Parasit. 57: 891-900, (1971). However, once merozoites produced by this method are released, the culture is terminated. It is current dogma that Eimeria exhibits synchronized growth and therefore can not be induced to produce an immortal line of merozoites, ie., a line of merozoites that does not terminate, after a relatively short period of time, in the release of oocysts.
Therefore, there remains a longfelt and longstanding need for an economical and efficient method to obtain sufficient Coccidia cells for use in vaccine production and other endeavers. Growth of merozoites, in vitro, on immortalized mammalian cell would be desirable. However, as noted above, no in vitro culture system, providing suitable host cells, has previously been available that allows for indefinite propagation of the asexual phase of Coccidia, ie, the merozoites, with conversion to the infectious stage that can be induced only as required.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application